Method for reducing angiogenesis by administration of a scatter factor inhibitor

ABSTRACT

This invention relates to a method of enhancing wound healing and to a method of enhancing organ transplantation utilizing scatter factor, either alone or in combination with a growth factor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/048,813, filed Mar. 26, 1998, which is a continuation-in-part of U.S.patent application Ser. No. 08/746,636, filed Nov. 13, 1996, now U.S.Pat. No. 5,837,676, which is a continuation of U.S. patent applicationSer. No. 08/138,667, filed Oct. 18, 1993, now abandoned, the contents ofeach of which are hereby incorporated by reference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under NIH Grant No.CA50516. As such, the government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to a method of enhancing wound healing and to amethod of enhancing organ transplantation comprising the administrationof scatter factor to promote angiogenesis.

BACKGROUND OF THE INVENTION

Scatter factor has previously been described as a cytokine which issecreted by fibroblasts (see Stroker et al., J. Cell Sci., Vol. 77, pp.209-223 (1985) and Stoker et al., Nature (London), Vol. 327, pp. 238-242(1987)) and by vascular smooth muscle cells (see Rosen et al., In VitroCell Dev. Biol., Vol. 25, pp. 163-173 (1989)). Scatter factor has beenshown to disperse cohesive epithelial colonies and stimulate cellmotility. In addition, scatter factor has been shown to be identical tohepatocyte growth factor (HGF) (see Weidner et al., Proc. Nat'l. Acad.Sci. USA, Vol. 88, pp. 7001-7005 (1991) and Bhargava et al., Cell GrowthDiffer., Vol. 3, pp. 11-20 (1992)), which is an independentlycharacterized serum mitogen (see Miyazawa et al., Biochem. Biophys. Res.Commun., Vol. 169, pp. 967-973 (1989) and Nakumura et al., Nature(London), Vol. 342, pp. 440-443 (1989)). Scatter factor induces kidneyepithelial cells in a collagen matrix to form branching networks oftubules, suggesting that it can also act as a morphogen (see Montesanoet al., Cell, Vol. 67, pp. 901-908 (1991)).

Scatter factor (HGF) is a basic heparin-binding glycoprotein consistingof a heavy (58 kDa) and a light (31 kDa) subunit. It has 38% amino acidsequence identity with the proenzyme plasminogen (see Nakumura et al.,Nature (London), Vol. 342, pp. 440-443 (1989)) and is thus related tothe blood coagulation family of proteases. Its receptor in epitheliumhas been identified as the c-met protooncogene product, a transmembranetyrosine kinase (see Bottaro et al., Science, Vol. 251, pp. 803-804(1991) and Naldini et al., Oncogene, Vol. 6, pp. 501-504 (1991)).

Scatter factor has been found to stimulate endothelial chemotactic andrandom migration in Boyden chambers (see Rosen et al., Proc. Soc. Exp.Biol. Med., Vol. 195, pp. 34-43 (1990)); migration from carrier beads toflat surfaces (see Rosen et al., Proc. Soc. Exp. Biol. Med., Vol. 195,pp. 34-43 (1990)); formation of capillary-like tubes (see Rosen et al.,Cell Motility Factors, (Birkhauser, Basel) pp. 76-88 (1991)) and DNAsynthesis (see Rubin et al., Proc. Nat'l Acad. Scil USA, Vol. 88, pp.415-419 (1991)). In addition, preliminary studies have suggested thatscatter factor induces endothelial secretion of plasminogen activators(see Rosen et al., Cell Motility Factors, (Birkhauser, Basel) pp. 76-88(1991)).

The term “angiogenesis”, as used herein, refers to the formation ofblood vessels. Specifically, angiogenesis is a multistep process inwhich endothelial cells focally degrade and invade through their ownbasement membrane, migrate through interstitial stroma toward anangiogenic stimulus, proliferate proximal to the migrating tip, organizeinto blood vessels, and reattach to newly synthesized basement membrane(see Folkman et al., Adv. Cancer Res., Vol. 43, pp. 175-203 (1985)).These processes are controlled by soluble factors and by theextracellular matrix (see Ingber et al., Cell, Vol. 58, pp. 803-805(1985)).

Because proteases, such as plasminogen activators (the endothelialsecretion of which is induced by scatter factor) are required during theearly stages of angiogenesis, and since endothelial cell migration,proliferation and capillary tube formation occur during angiogenesis,the inventors hypothesized that scatter factor might enhance angiogenicactivity in vivo. In addition, it is desirable to enhance angiogenicactivity so that would healing and organ transplantation can beenhanced.

It is therefore an object of this invention to provide a method ofenhancing angiogenic activity.

It is a further object of this invention to provide a method ofenhancing wound healing.

It is a still further object of this invention to provide a method ofenhancing organ transplantation.

SUMMARY OF THE INVENTION

This invention is directed to a method of promoting angiogenesis byadministration of scatter factor. The promotion of angiogenesis can beused for promoting would healing and treating various conditions wherethe promotion of angiogenesis is desirable, including, but not limitedto, ischemia.

BRIEF DESCRIPTION OF THE DRAWINGS

The above brief description, as well as further objects and features ofthe present invention, will be more fully understood by reference to thefollowing detailed description of the presently preferred, albeitillustrative, embodiments of the present invention when taken inconjunction with the accompanying drawing wherein:

FIG. 1 is comprised of FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D. FIG. 1represents the result of a murine angiogenesis assay. FIG. 1A showsMATRIGEL plugs (arrowheads) before excision of the plugs. FIG. 1B showsplugs (arrowheads) after excision of the plugs. FIG. 1C shows thequantification by digital image analysis for athymic mice. FIG. 1D showsthe quantitation by digital image analysis for C57BL mice;

FIG. 2 is comprised of FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D. FIG. 2represents the microscopic appearance of MATRIGEL plugs. FIG. 2Arepresents sections of plugs from athymic control (0 ng scatter factor)mice. FIG. 2B represents sections of plugs from athymic mice whichcontain 2 ng scatter factor. FIG. 2C represents sections of plugs fromathymic mice which contain 20 ng scatter factor. FIG. 2D representssections of plugs from athymic mice which contain 200 ng of scatterfactor;

FIG. 3 is comprised of FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D and FIG. 3E.FIG. 3 represents scatter factor-induced angiogenesis in rat corneas. Asshown in FIG. 3A, no angiogenic response was observed in control pelletscontaining PBS. As shown in FIG. 3B, the response for 50 ng scatterfactor was positive but weak in comparison with high concentrations ofscatter factor. As shown in FIG. 3C, and FIG. 3D, scatter factorconcentrations of 100 ng and 500 ng, respectively, gave strong positiveresponses. FIG. 3E shows a strong angiogenic response which was inducedby 150 ng of basic FGF, a positive control;

FIG. 4 is comprised of FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D and FIG. 4E.FIG. 4 represents stimulation of plasminogen activator expression byscatter factor. FIG. 4A shows secreted activity during a 6 hourcollection interval. FIG. 4B shows secreted activity intracellularly.FIG. 4C shows total (secreted plus intracellular) activity. FIG. 4D andFIG. 4E show plasminogen activator activity in medium and in lysatesfrom cells treated with scatter factor at 20 ng/ml assayed in thepresence of goat anti-human urokinase IgG (auPA), goat anti-human tissuetype PA IgG (atPA) or goat nonimmune IgG (NI IgG) (200 mg/ml),respectively.

FIG. 5 is comprised of FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D. FIG. 5represents the results of immunohistochemical staining of skin biopsysamples for scatter factor. FIG. 5A, FIG. 5B and FIG. 5C showimmunohistochemical staining of psoriatic plaques. FIG. 5D showsimmunohistochemical staining of normal skin from a patient withpsoriasis.

FIGS. 6A and 6B. Production of SF by SF-transfected and control MDAMB231cells. FIG. 6A: Western blotting. One-hundred μl aliquots ofconcentrated conditioned media (see Materials and Methods) wereelectrophoresed on a 12% non-reduced SDS-polyacrylamide gel and blottedto detect SF. Lanes 1-4 represent media from (SF+neo) transfected clones21, 29, 52, and 3-7, respectively; lanes 5 and 6 represent media fromneo-only transfected clones 32 and 34; and lanes 7 and 8 representpositive standards [native human placental SF and rhSF (50 ng)]. FIG.6B. Northern blotting. Total cell RNA was isolated from twoSF-transfected clones (21 and 29) and one control (neo) clone (34).Equal aliquots of RNA (30 μg/lane) were electrophoresed, and blots wereprepared. Blots were probed using a 500 bp SF cDNA probe labeled withdigoxigenin and developed using anti-digoxigenin antibody. Bands of theexpected size [slightly larger than full-length SF cDNA (2.3 kb)] wereobserved for clones 21 and 29, whereas no bands were detected for clone34.

FIG. 7. Expression of SF and c-met mRNA in MDAMB231 cell clones byRT-PCR analysis. The 435 bp SF reaction product was detected in SFtransfected clone 21 (lane 1) and 29 (lane 2), but not in controltransfected clone 32 (lane 3) and 34 (lane 4). However, the 655 bp c-metreaction product and the 764 bp β-actin product were detected in allfour clones.

FIG. 8. In vitro proliferation kinetics of scatter factor (SF)transfected and control MDAMB231 human mammary carcinoma cell clones.Sixty thousand cells/well were seeded into 12-well dishes on day −1 andallowed to overnight in 1.0 ml of DMEM plus 5% serum. Medium wasreplaced on day 0 and on the days indicated, and wells were counted induplicate by hemacytomer, as indicated. Each point is a mean ofduplicate assays, with a range of ±10% of the mean value.

FIGS. 9A and 9B. In vivo tumor growth of SF-transfected and controlclones of MDAMB231 human mammary carcinoma cells in the mammary fat padsof nude mice. Cells were injected at time 0 into the mammary fat pads,and tumor volumes were determined each week from measurements of threeperpendicular diameters. [See Materials and Methods for details,including numbers of cells and animals injected.] FIGS. 9A and 9B showresults from two separate experiments (Experiments 3 and 2,respectively). Comparisons of pooled data for SF+ clones (21+29) vs SF−clones (32+34) were made at each time point using two-tailed t-tests.Significant comparisons are shown in the figure (* indicates P<0.05; **indicates P<0.01). In addition, in FIG. 9A, comparisons of clone 21 vspooled clones (32+34) were significant at P<0.01 for all time points(wks 1-9); and comparisons of clone 29 vs pooled clones (32+34) weresignificant at P<0.01 for wks 7-9.

FIGS. 10A-10E. Human dermal microvascular endothelial cell (HDMEC)chemotaxis assays. FIG. 10A: Stimulation of HDMEC chemotaxis by SF andits inhibition by anti-SF monoclonal AB. rhSF was tested for chemotacticactivity alone or in the presence of anti-SF monoclonal MO294 (AB) at 2or 20 μg/ml (following a 30 min pre-incubation with AB). Values plottedare means±SEMs of three assays. FIGS. 5B and 5C: Assays of conditionedmedia (CM) from SF+ vs SF− cell clones. In FIG. 10B, CM was concentrated10-fold using a 10 kDa membrane and assayed for chemotactic activity atthree different protein concentrations. Values plotted are means oftriplicate assays. SEMs were <5% of the mean values. In FIG. 10C, CM (50μg/ml) was preincubated without or with anti-SF monoclonal AB (20 μg/ml)for 30 min and assayed for chemotactic activity. Migration values areexpressed as a percentage of the control (no CM, no AB) and representmeans±SEMs of triplicate assays. * indicates significant inhibition ofmigration by AB (P<0.001). FIGS. 10D and 10E: Assays of extracts ofprimary tumors derived from SF+ vs SF− clones. In FIG. 10D, tumorextracts were tested for chemotactic activity at three different proteinconcentrations. For each clone, two different extracts were tested, andeach extract was assayed in triplicate. Migration values for the twoextracts from the same clone were nearly identical, and these valueswere averaged. Thus, each value plotted is a mean of six assays perclone, with SEMs<5% of means. In FIG. 10E, extracts from clones 21 and29 (50 μg/ml) were pre-incubated without or with anti-SF monoclonal AB(20 μg/ml) for 30 min and assayed for chemotactic activity. Values areexpressed as a percentage of the control (no extract, noAB) andrepresent means±SEMs of triplicate assays. * indicates significantinhibition of migration by AB (P<0.01).

FIGS. 11A-11E. Angiogenic activity of primary tumor extracts from SF+and SF− tumors in the rat cornea. A 5 μl pellet of Hydron containingtest samples was inserted into the avascular rat cornea about 1-1.5 mmfrom the limbus. After 7 days, corneas wee perfused with colloidalcarbon, and whole mount preparations were photographed. FIG. 11A shows anegative neovascular response 7 days after implanting a Hydron pelletcontaining buffered saline. FIG. 11B shows a positive neovascularresponse induced by 50 ng of human recombinant SF. Note the directionalingrowth of capillaries toward the Hydron implant located at the centerof the photograph. [The dose of 50 ng of SF gives a strong butsub-maximal angiogenic response; SF doses of 100 ng or more give maximalresponses (Grant et al., 1993).] A vigorous neovascular response wasinduced by 5 μg of extract from a SF+ clone 21 tumor (FIG. 11C). Apositive but substantially weaker neovascular response was induced by 5μg of SF− clone 32 tumor extract (FIG. 11D). FIG. 11E shows a markedlyattenuated neovascular response induced by 5 μg of SF+ clone 21 tumorextract in the presence of 2.5 μg of anti-SF neutralizing monoclonalantibody MO294.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of promoting angiogenesisin a tissue or subject by administering scatter factor to a subject inneed of angiogenesis promotion. Specifically, the method provided by thepresent invention involves the administration of scatter factor topromote angiogenesis in various tissues to promote wound healing.

The administration of scatter factor may be effected by administrationof the protein itself or administration of a nucleic acid encodingscatter factor by the use of standard DNA techniques.

Scatter factor protein may be administered to a tissue or subjecttopically or by intravenous, intramuscular, intradermal, subcutaneous orintrapertioneal injection. Scatter factor protein is administered inamounts sufficient to promote angiogenesis in a subject, which is in theamount of about 0.1-1000 ng/kg body weight.

Scatter factor protein may be administered as the wild type scatterfactor protein, or analogues thereof, and may be produced syntheticallyor recombinantly, or may be isolated from native cells. As used herein,“analogue” means functional variants of the wild type protein, andincludes scatter factor protein isolated from mammalian sources otherthan human, such as mouse, as well as functional variants thereof.

A nucleic acid sequence encoding scatter factor administered to a mammalmay be genomic DNA or cDNA. The nucleic acid sequence may beadministered using a number of procedures known to one skilled in theart, such as electroporation, DEAE Dextran, monocationic liposomefusion, polycationic liposome fusion, protoplast fusion, DNA coatedmicroprojectile bombardment, by creation of an in vivo electrical field,injection with recombinant replication-defective viruses, homologousrecombination, and naked DNA transfer. It is to be appreciated by oneskilled in the art that any of the above methods of DNA transfer may becombined.

A nucleic acid encoding scatter factor may also be administered to amammal using gene therapy, i.e. by the administration of a recombinantvector containing a nucleic acid sequence encoding scatter factor. Thenucleic acid sequence may be, for example, genomic DNA or cDNA. Therecombinant vector containing nucleic acid encoding scatter factor maybe administered to a mammal using any number of procedures known to oneskilled in the art, including, but not limited to, electroporation, DEAEDextran transfection, calcium phosphate transfection, cationic liposomefusion, protoplast fusion, by creation of an in vivo electrical field,DNA coated microprojectile bombardment, injection with recombinantreplication-defective viruses, homologous recombination, gene therapy,and naked DNA transfer. It is to be appreciated by one skilled in theart that any of the above methods of nucleic acid transfer may becombined. Accordingly, a cell, such as a stem cell or a tumor cell whichexpresses scatter factor introduced therein through viral transducerion,homologous recombination, or transfection is also provided by thepresent invention. This call may then be administered to a subject topromote angiogenesis.

The recombinant vector may comprise a nucleic acid of or correspondingto at least a portion of the genome of a virus, where this portion iscapable of directing the expression of a nucleic sequence encodingscatter factor, operably linked to the viral nucleic acid and capable ofbeing expressed as a functional gene product in the subject mammal. Therecombinant vectors may be derived from a variety of viral nucleic acidsknown to one skilled in the art, e.g. the genomes of HSV, adenovirus,adeno-associated virus, Semiliki Forest virus, vaccinia virus, and otherviruses, including RNA and DNA viruses.

The recombinant vectors may also contain a nucleotide sequence encodingsuitable regulatory elements so as to effect expression of the vectorconstruct in a suitable host cell. As used herein, “expression” refersto the ability of the vector to transcribe the inserted DNA sequenceinto mRNA so that synthesis of the protein encoded by the insertednucleic acid can occur. Those skilled in the art will appreciate that avariety of enhancers and promoters are suitable for use in theconstructs of the invention, and that the constructs will contain thenecessary start, termination, and control sequences for propertranscription and processing of the nucleic acid sequence encodingscatter factor when the recombinant vector construct is introduced intoa mammal.

Vectors suitable for the expression of the nucleic sequence encodingscatter factor are well known to one skilled in the art and includepMEX, pRSX24 (provided by Dr. George Vande Woude, Frederick CancerCenter, Frederick, Md.), pSV2neo (Clonetech), pET-3d (Novagen), pProEx-1(Life Technologies), pFastBac 1 (Life Technologies), pSFV (LifeTechnologies), pcDNA II (Invitrogen), pSL301 (Invitrogen), pSE280(Invitrogen), pSE380 (Invitrogen), pSE420 (Inventrogen), pTrcHis A,B,C(Invitrogen), pRSET A,B,C (Invitrogen), pYES2 (Invitrogen), pAC360(Invitrogen), pVL1392 and pVl1392 (Invitrogen), pCDM8 (Invitrogen),pcDNA I (Invitrogen), pcDNA I(amp) (Invitrogen), pZeoSV (Invitrogen),pcDNA 3 (Invitrogen), pRc/CMV (Invitrogen), pRc/RSV (Invitrogen), pREP4(Invitrogen), pREP7 (Invitrogen), pREP8 (Invitrogen), pREP9(Invitrogen), pREP10 (Invitrogen), pCEP4 (Invitrogen), pEBVHis(Invitrogen), and λPop6. Other vectors would be apparent to one skilledin the art.

Suitable promoters include, but are not limited to, constitutivepromoters, tissue specific promoters, and inducible promoters.Expression of the nucleic acid sequence encoding scatter factor may becontrolled and affected by the particular vector into which the nucleicacid sequence has been introduced. Some eukaryotic vectors have beenengineered so that they are capable of expressing inserted nucleic acidsto high levels within the target cell. Such vectors utilize one of anumber of powerful promoters to direct the high level of expression.Eukaryotic vectors use promoter-enhancer sequences of viral genes,especially those of tumor viruses. A particular embodiment of theinvention provides for regulation of expression of the nucleic acidsequence encoding scatter factor using inducible promoters. Non-limitingexamples of inducible promoters include, but are not limited to,metallothionine promoters and mouse mammary tumor virus promoters.Depending on the vector, expression of the nucleic acid sequenceencoding scatter factor would be induced in the mammal by the additionof a specific compound at a certain point in the growth cycle of thecells of the mammal. Other examples of promoters and enhancers effectivefor use in the recombinant vectors include, but are not limited to, CMF(cytomegalovirus), SV40 (simian virus 40), HSV (herpes simplex virus),EBV (epstein-barr virus), retroviral, adenoviral promoters andenhancers, and tumor cell specific promoters and enhancers.

It is within the confines of the invention that scatter factor may beadministered in combination with a growth factor to promoteangioogenesis, including, but not limited to TGF-α, FGF and PDGF.

Scatter factor, in the form of a protein, nucleic acid, or a recombinantvector containing nucleic acid encoding scatter factor, may beadministered to a subject prior to, simultaneously with or subsequent toadministration of a growth factor.

For the purposes of gene transfer into a tissue or subject, arecombinant vector containing nucleic acid encoding scatter factor maybe combined with a sterile aqueous solution which is preferably isotonicwith the blood of the recipient. Such formulations may be prepared bysuspending the recombinant vector in water containing physiologicallycompatible substances such as sodium chloride, glycine, and the like,and having buffered pH compatible with physiological conditions toproduce an aqueous solution, and rendering such solution sterile. In apreferred embodiment of the invention, the recombinant vector iscombined with a 20-25% sucrose in saline solution in preparation forintroduction into a mammal.

The amounts of nucleic acid encoding scatter factor, or nucleic acidencoding scatter factor contained in a vector are administered in amounts sufficient to promote angiogenesis in a subject. However, theexact dosage will depend on such factors as the purpose ofadministration, the mode of administration, and the efficacy of thecomposition, as well as the individual pharmacokinetic parameters of thesubject. Such therapies may be administered as often as necessary andfor the period of time as judged necessary by one of skill in the art.

Non-limiting examples of tissues into which nucleic acid encodingscatter factor may be introduced to promote angiogenesis includefibrous, endothelial, epithelial, vesicular, cardiac, cerebrovascular,muscular, vascular, transplanted, and wounded tissues.

Transplanted tissues are, for example, heart, kidney, lung, liver andocular tissues.

The tissues into which nucleic acid encoding scatter factor may beintroduced to promote angiogenesis include those associated withdiseases or conditions selected from the group consisting of ischemia,circulatory disorders, vascular disorders, myocardial ischemicdisorders, myocardial disease, pericardial disease or congenital heartdisease. Non-limiting examples of ischemia are cyocardial ischemia,cerebrovascular ischemia and veno-occlusive disorder. An example ofmyocardial ischemia and veno-occlusive disorder. An example ofmyocardial ischemia is coronary artery disease.

In further embodiments of the invention, scatter factor is used toenhance would healing, organ regeneration, and organ transplantation,including the transplantation of artificial organs. In addition, scatterfactor can be used to accelerate endothelial cell coverage of vasculargrafts in order to prevent graft failure due to reocclusion, and toenhance skill grafting. Further, antibodies to scatter factor can beused to treat tumors and to prevent tumor growth.

Murine Angiogenesis Assays. In order to perform the murine angiogenesisassay, angiogenesis was assayed as growth of blood vessels fromsubcutaneous tissue into a solid gel of basement membrane containing thetest sample. MATRIGEL (7 mg in 0.5 ml; Collaborative Research), inliquid form at 4° C. was mixed with scatter factor and injected into theabdominal subcutaneous tissues of athymic XID nude beige mice or C57BL/6mice. MATRIGEL rapidly forms a solid gel at body temperature, trappingthe factor to allow slow release and prolonged exposure to surroundingtissues. After 10 days, the mice were sacrificed and the MATRIGEL plugswere excised and fixed in 4% formaldehyde in phosphate buffer. Plugswere embedded in paraffin, sectioned, stained with Masson's trichrome(which stains endothelial cells reddish-purple and stains the MATRIGELviolet or pale green), and examined for ingrowth of blood vessels.Vessel formation was quantitated from stained sections using the OPTIMAXdigital image analyzer connected to an OLYMPUS microscope (see Grant etal., Cell, Vol. 58, pp. 933-943 (1989)). Results were expressed as meanvessel area per field±SEM (arbitrary units) or as total vessel area(mm²) in 20 random fields.

EXAMPLE I

Scatter Factor Preparations. In order to prepare scatter factorpreparations, mouse scatter factor was purified from serum-free culturemedium from ras-transformed NIH/23T3 cells (clone D4) by cation-exchangechromatography as described by Rosen et al., Proc. Soc. Exp. Biol. Med.,Vol. 195, pp. 34-43 (1990), followed by immunoaffinity chromatographyand ultrafiltration. Recombinant human HGF (rhHGF) was provided byToshikazu Nakamura (Kyushu University, Fukuoka, Japan). Scatter factor(HGF) is commercially available from Collaborative Research, Bedford,Mass.

Antibody Preparations. In order to make the antibody preparations,antisera to native human placental scatter factor and rhHGF wereprepared by immunizing rabbits with purified factors (see Bhargava etal., Cell Growth Differ., Vol. 3, pp. 11-20 (1992) and Bhargava et al.,Cell Motility Factors, (Birkhauser, Basel) pp. 63-75 (1991)). A chickenegg yolk antibody to human placental scatter factor was prepared byimmunizing two White Leghorn hens, 22-24 weeks old, with 500 mg of humanplacental scatter factor emulsified in complete Freund's adjuvant.Booster injections were given 14 to 28 days later, and the eggs werecollected daily. The IgG fraction from seven eggs was extracted andpartially purified by the methods described by Polson et al., Immunol.Commun., Vol. 9, pp. 495-514 (1980). The final preparation contained 80mg of protein per ml in phosphate-buffered saline (PBS). Antibodyspecificity was established by recognition of mouse and human scatterfactors on immunoblots, specific binding of scatter factor toantibody-Sepharose columns, and inhibition of the in vitro biologicactivities of mouse and human scatter factor.

Plasminogen Activator Assays. In order to perform plasminogen activatorassays, bovine brain microvessel endothelial cells (BBEC) were isolatedfrom brain cortex after removal of the pia mater, identified asendothelial, and cultured by standard techniques. BBEC (passage 10-12)at about 80% confluency in 60 mm Petri dishes were treated with mousescatter factor for 24 hours, washed, and incubated for 6 hours in 2.5 mlof serum-free Dulbecco's modified Eagle's medium (DMEM) to collectsecreted proteins. The cells were washed, scraped into PBS, collected in0.5 ml of PBS by centrifugation, and lysed by sonication. Aliquots ofmedium and cell lysates were assayed for PA activity by a two-stepchromogenic reaction as described by Coleman et al., Ann. N.Y. Acad.Sci., Vol. 370, pp. 617-626 (1991). Human high molecular weighturokinase (American Diagnostica, Greenwich, Conn.) was used as thestandard. The protein content of the lysate was determined by using theBradford dye-binding assay (Bio-Rad).

Murine Angiogenesis Assays. In order to perform the murine angiogenesisassay, angiogenesis was assayed as growth of blood vessels fromsubcutaneous tissue into a solid gel of basement membrane containing thetest sample. Matrigel (7 mg in 0.5 ml; Collaborative Research) in liquidform at 4° C. was mixed with scatter factor and injected into theabdominal subcutaneous tissues of athymic XID nude beige mice or C57BL/6mice. Matrigel rapidly forms a solid gel at body temperature, trappingthe factor to allow slow release and prolonged exposure to surroundingtissues. After 10 days, the mice were sacrificed and the Matrigel plugswere excised and fixed in 4% formaldehyde in phosphate buffer. Plugswere embedded in paraffin, sectioned, stained with Masson's trichrome(which stains endothelial cells reddish-purple and stains the Matrigelviolet or pale green), and examined for ingrowth of blood vessels.Vessel formation was quantitated from stained sections using the Optimaxdigital image analyzer connected to an Olympus microscope (see Grant etal., Cell, Vol. 58, pp. 933-943 (1989)). Results were expressed as meanvessel area per field±SEM (arbitrary units) or as total vessel area(mm²) in 20 random fields.

Rat Cornea Angiogenesis Assays. In order to perform the rat corneaangiogenesis assay, angiogenesis was assayed in the avascular ratecornea, as described by Polverini et al., Lab. Invest., vol. 51, pp.635-642 (1984). Test samples were combined 1:1 with a sterile solutionof Hydron (Interferon Laboratories, New Brunswick, N.J.) and air-driedovernight. A 5 ml pellet was inserted into a surgically created pocketin the corneal stroma and positioned 1-1.5 mm from the limbus. Corneaswere examined daily with a dissecting microscope for up to 7 days forcapillary growth. Assay responses were scored as positive if sustaineddirectional ingrowth of capillary sprouts and hairpin loops occurredduring the observation period. Responses were scored as negative eitherwhen no neovascularization was detected or when only an occasionalsprout or hairpin loop was observed that showed no evidence of sustaineddirectional ingrowth. After 7 days, corneas were perfused with colloidalcarbon, and whole-mount preparations were examined and photographed.

Immunohistochemistry. To study immunohistochemistry,five-micrometer-thick cryostat sections were prepared from biopsysamples of plaques or of areas of normal skin in patients with activepsoriasis. The sections were sustained by using an avidin-biotinimmunoperoxidase technique (see Griffiths et al., Am. Acad. Dermatol.,Vol. 20, pp. 617-629 (1989)). The chromogen was Texas red conjugated toavidin. The primary antibody was rabbit polyclonal antiserum to purifiednative human placental scatter factor or to rhHGF (1:1000 dilution).Nonimmune rabbit serum (1:1000) was used as a negative control.

In vivo Assays. Two different in vivo assays were used to evaluate theangiogenic activity of mouse scatter factor. In the first assay, themurine angiogenesis assay, samples mixed with MATRIGEL, a matrix ofreconstituted basement membrane, were injected subcutaneously into mice.After 10 days, the mice were sacrificed for histologic and morphometricanalysis of MATRIGEL plugs. Control plugs were found to be pale pink,while plugs containing scatter factor were found to be bright red andoften contained superficial blood vessels (see FIG. 1A and FIG. 1B).

Histologic analysis showed little cellularity in control plugs (see FIG.2A). Plugs containing 2 ng of scatter factor often had increased numbersof cells (see FIG. 2B), 90% of which stained for factor VIII antigen, anendothelial cell marker (not shown). At 20 ng of scatter factor, cellnumber was increased, and vessels were present (FIG. 2C). At 200 ng ofscatter factor, plugs were even more cellular, with endothelial cellsmaking up 50-60% of the cell population. Many large vessels containingsmooth muscle cells were seen (see FIG. 2D).

Morphometric analysis of vessel area (see Grant et al., Cell, Vol. 58,pp. 933-943 (1989)) revealed a dose-dependent angiogenic response inathymic (FIG. 1C) and C57BL (FIG. 1D) mice, with half-maximal andmaximal responses at about 20 and 200 ng, respectively. Histologicexamination at day 10 showed no evidence of inflammation in scatterfactor-containing plugs in athymic mice. In C57BL, no inflammation wasobserved at ≦200 ng of scatter factor, but leukocytic infiltration waspresent in tissue surrounding the plugs at ≦2000 ng of scatter factor.

In the second assay, samples were implanted in the avascular rat corneato allow ingrowth of blood vessels from the limbus. Control implantsgave no positive responses (see Table 1, below, and FIG. 3A), whileimplants containing mouse scatter factor induced a dose-dependentcorneal neovascularization. Responses at 50 ng (FIG. 3B) were reduced inintensity compared with those at 100 and 500 ng (FIG. 3C and FIG. 3D,respectively). The maximal response to scatter factor was observed atdoses of ≦100 ng and was similar to the response to 150 ng of humanbasic FGF, a positive control (see FIG. 3E).

TABLE 1 Neovascular responses induced in rat corneas by scatter factor(SF) Corneal neovascularization Positive Content of pellet responses %Negative controls Sham implant 0/3 0 Hydron 0/2 0 PBS 0/2 0 Positivecontrol Basic FGF (150 ng) 4/4 100 scatter factor (SF)   5 ng 0.4 0  50ng  3/5* 60  100 ng 5/5 100  500 ng 5/5 100 1000 ng  5/5+ 100 *Responseswere much weaker in intensity compared with implants containing 100 or500 ng of scatter factor. +Corneas showed significant inflammation.

rhHGF also induced angiogenesis in the rat cornea (see Table 2, below).At 100 ng, positive responses were observed in four of five implants. At500 ng of rhHGF, all all implants gave positive responses. Chicken andrabbit antibodies to human placental scatter factor strongly inhibitedthe angiogenic responses to mouse scatter factor and rhHGF, but not tobasic FGF (see Table 2).

TABLE 2 Neovascular responses induced in rat corneas by native mousescatter factor (SF) and rhHGF with or without antibody (Ab) Cornealneovascularization Positive Content of pellet responses % ControlsHydron + PBS 0/8 0 Chicken Ab 0/4 0 Rabbit Ab (Ab 978) 0/3 0 Basic FGF(150 ng) 3/3 100 Basic FGF (150 ng) + rabbit Ab 3/3 100 Factor ± AbMouse SF (100 ng) 3/3 100 Mouse SF (100 ng) + chicken Ab  1/5* 20 rhHGF(100 ng) 4/5 80 rhHGF (500 ng)  5/5+ 100 rhHGF (100 ng) + chicken Ab 2/5* 33 rhHGF (100 ng) + rabbit Ab 0/5 0 Antibodies were diluted inPBS. Final dilutions after mixing with Hydron were 1:20 for the chickenantibodies and 1:20 for the rabbit antibodies. *Responses scored aspositive were very weak. +This concentration of rhHGF was inflammatory.

To asses inflammation, corneas wee examined by direct stereomicroscopydaily for the duration of the experiments. Corneas chosen at random wereexamined histologically at 6, 12 and 24 hours and at 3, 5, and 7 daysafter implantation of scatter factor and control pellets. Inflammationwas not detected at lower angiogenic doses of scatter factor (50-500 ngof mouse scatter factor, 100 ng of rhHGF). At higher doses (50-500 ng ofmouse scatter factor, 500 ng of rhHGF), a prominent inflammatoryinfiltrate was observed. The majority of cells were monocytes andmacrophages, as judged by morphology and immunostaining for F4/80, amacrophage/monocyte marker.

Plasminogen activators convert plasminogen into plasmin, a potent serineprotease that lyses fibrin clots, degrades components of extracellularmatrix, and activates enzymes (e.g., procollagenases) that furtherdegrade matrix (see Saksela et al., Anu. Rev. Cell Biol., Vol. 4, pp.93-126 (1988)). The inventors have discovered that scatter factorinduces large dose-dependent increases in secreted (see FIG. 4A) andcell-associated (see FIG. 4B) plasminogen activator activity inmicrovascular endothelium (BBEC). Total plasminogen activator activity(secretec plus cell-associated) was increased 4-fold relative to controlwhen scatter factor was present at 20 ng/ml (Z0.2 nM). Similar resultswere obtained in large vessel endothelium (not shown). Most of thesecreted and cell-associated plasminogen activator activity in BBEC wasblocked by antibodies to urokinase, but not by antibodies to tissueplasminogen activator (see FIG. 4D to FIG. 4E).

Angiogenesis is often associated with chronic inflammation diseases.Psoriasis is a common inflammatory skin disease characterized byprominent epidermal hyperplasia and neovascularization in the dermalpapillae. Frozen sections of biopsy samples from psoriatic plaques from10 patients each showed positive immunohistochemical staining forscatter factor in spindle-shaped and mononuclear cells within the dermalpapillae and papillary dermis. Antisera to human placental scatterfactor and rhHGF gave an identical staining pattern, as illustrated inFIG. 5A. Scatter factor-positive cells were arranged in a perivasculardistribution. Cells of the blood vessel wall did not stain for scatterfactor (see FIG. 5C). Normal skin from psoriasis patients or from normalsubjects showed little or no staining for scatter factor (FIG. 5D).Sections from psoriatic plaques treated with nonimmune serum as theprimary antibody (negative control) showed no staining (FIG. 5B).

Hence, the inventors have determined that physiologic quantities ofscatter factor [100-200 ng (Z1-2 pmol)] induced strong angiogenicresponses in two in vivo assays. It is likely that this angiogenicactivity is due, in part, to direct effects on endothelium since: (i)scatter factor stimulates endothelial migration, proliferation, and tubeformation in vitro; (ii) histologic studies showed no evidence ofinflammation at scatter factor doses that gave strong angiogenicresponses; and (iii) anti-scatter factor antibodies blocked theangiogenic responses. The inventors also found that scatter factorstimulates endothelial cell expression of urokinase. Urokinase, bound toits specific cells surface receptor, is thought to mediate focal,directed, extracellular proteolysis, which is required for endothelialcell invasion and migration during the early stages of angiogenesis.

Growth factors TGFβ, FGF, and platelet-derived growth factor (PDGF) arepresent in MATRIGEL and in the matrices of several tissues, includingthe cornea. The inventors have discovered that combinations of scatterfactor and either TGFβ, FGF, or PDGF provide greater stimulation ofendothelial tube formation in vitro than did the same agents usedindividually. The concentrations studied (1 ng/ml) were about 10 timesthose found in 250 mg of MATRIGEL, and scatter factor stronglystimulated tube formation on its own, by up to 8 times the amountstimulated by the control.

The major scatter factor producer cells are fibroblasts, smooth musclecells, and leukocytes. With rare exceptions, responder cells(epithelium, endothelium, melanocytes) are nonproducers. Theimmunohistochemical studies of psoriatic plaques suggest that scatterfactor is produced by cells located outside of the blood vessel wall.Studies by the inventors have indicated that cultured endothelial cellsexpress c-met mRNA and that immunoreactive c-met protein is present inblood vessel wall cells (endothelium and pericytes) in psoriaticplaques. This suggests that scatter factor may play a role inmicrovessel formation or elongation in psoriasis and that its likelymode of action is paracrine.

Scatter factor (HGF) stimulates motility, invasiveness, proliferation,and morphogenesis of epithelium, and it may be involved in physiologicand pathologic processes such as embryogenesis, wound healing, organregeneration, inflammation, and tumor invasion. Angiogenesis is acomponent of each of these processes. Therefore, the in vivo biologicaction of scatter factor may be due, in part, to its effects on bothepithelial and vascular endothelial cells.

EXAMPLE II

I. Materials and Methods

Cell Lines, Sources, and Culture. MDAMB231 human breast cancer cells andMadin-Darby canine kidney (MDCK) epithelial cells were obtained from theAmerican Type Culture Collection (ATCC), Manassas, Va. Cells werecultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with10% (v/v) fetal calf serum, L-glutamine (5 mM), nonessential amino acids(0.1 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and fungizone(0.25 μg/ml) (all from Bio Whittaker, Walkersville, Md.). Cells weresubcultured weekly as described earlier (Rosen et al., Cancer Res57:5315-5321 (1991)). Human dermal microvascular endothelial cells(HDMECs) were purchased from Clonetics, Inc. (San Diego, Calif.) andcultured in EBM Bullet Kit medium (Clonetics), as per the manufacturer'sinstructions.

Transfection Vectors and DNA Transfection Method. MDAMB231 cells weretransfected with human SF cDNA using the vectors and method describedearlier (Rong et al., Mol Cell Biol 12: 5152-5158 (1992)). Briefly, theplasmid pRSX24, which was constructed by ligating full-length 2.3 kb SFcDNA into the BamHI-KpnI site of the pMEX vector, was provided by Dr.George Vande Woude (Frederick Cancer Center, Frederick, Md.). Cells wereeither: (a) co-transfected with pRXS24 plus pSV2neo (Clonetech) (whichcontains the neomycin-resistance gene); or (b) transfected with pSV2neoalone (control). The calcium phosphate method of DNA transfection wasused, exactly as described before (Rong et al., Mol Cell Biol 12:5152-5158 (1992)). Selection was carried out in culture mediumcontaining 0.4 mg/ml of G418 (Geneticin, GIBCO, Gaithersburg, Md.).Colonies of cells were trypsinized within cloning rings, transferred to24-well dishes, grown to confluency, and screened for release of SF intoserum-free DMEM by ELISA (see below). (SF+neo) clones that produced SFand a number of SF− (neo only) clones were expanded to 12-well and then100 mm dishes, grown up in the absence of G418, split once, and frozenin liquid nitrogen. (SF+neo) clones that produced the same high levelsof SF over multiple (>5) passages were designated SF+ permanenttransfectants; and they, along with several SF− (neo only) clones thatappeared to proliferate at roughly the same rate were chosen for furtherstudy. SF+ and SF− clones were frozen at multiple passages, but only theearliest passage cells were used for injection into animals.

Analysis of Transfected Clones for SF Expression. ELISA Screening ofConditioned Medium (CM). To generate CM, confluent cultures oftransfected clones were incubated in serum-free DMEM (0.1 ml/cm²) for 24hr at 37° C. CM sample were centrifuged (3000 RPM×20 min) to removedebris and stored at −80° C. until the time of assay. Samples wereassayed by our standard two-antibody “sandwich” ELISA, as describedearlier (Joseph et al., Natl Cancer Inst 87: 372-377 (1995); Rosen etal., J Cell Biol 127: 1783-1787 (1994)). The linear detection range isusually about 0.2 to 4.0 ng/ml of standard [recombinant human SF (rhSF),Genentech, Inc., South San Francisco, Calif.]. The assay has been shownto be specific for SF, since plasminogen (closely related to SF),albumin, and a variety of growth factors and cytokines are not detected.

Western Blotting of CM. Twenty-four hour CM samples (see above) fromtransfected clones were concentrated 10-fold with an Amicon Centricon-10concentrator (10 kDa cutoff), and 100 μl aliquots were electrophoresedon a 12% non-reduced SDS-polyacrylamide gel. Blots were probed usingmouse anti-human SF monoclonal antibody 23C2 (1:2000 dilution ofascites), and bound primary antibody was detected by detected byelectrochemiluminescence (ECL) (Amersham), as described before (Rosen etal., J Cell Biol 127: 225-234 (1994)). Recombinant human SF (Genentech)and native human placental SF (Grant et al., PNAS USA 90: 1937-1941(1993)) were utilized as standards.

Northern Blotting. Probe: A 565 bp portion of human SF cDNA cloned intothe BamHI-XhoI fragment of pBluescriptIIKS was provided by Dr. MoragPark (McGill University, Montreal, Canada). The SF cDNA fragment wasexcised using BamHI and XhoI and labelled with digoxigenin-11-dUTP bythe random prime method, using the Genius System DNA Labelling andDetection Kit (Boehringer Mannheim). RNA Isolation, Electrophoresis,Hybridization. Total cell RNA was isolated from near confluent cellcultures by acid guanidinium isothiocyanate-phenol-chloroformextraction. Equal aliquots of RNA (30 μg/lane) were electrophoresedthrough a 17% formaldehyde-1% agarose gel; transferred to a Nytranmembrane overnight in 10XSSC; and UV cross-linked. Prehybridization wascarried out for 1 hr at 50° C.; and hybridization was carried out in thesame buffer containing 50 ng/ml of probe, overnight, at 50° C. Membraneswere washed as follows: 2XSSC, 0.1% SDS, 10 min, 25° C. (twice); and0.1XSSC, 0.1% SDS, 30 min, 55° C. (twice). Detection: Hybrids weredetected using anti-digoxigenin antibody-alkaline phosphatase and analkaline phosphatase chemiluminescent reaction (Boehringer Mannheim),according to the manufacturer's instructions.

PCR Analysis. Expression of SF and c-met mRNA in SF-transfected andcontrol-transfected cells was assayed by RT-PCR analysis. Total cellularRNA (1 μg) was reverse transcribed using 10,000 U/ml Superscript RNaseH⁻Moloney murine leukemia virus reverse transcriptase (GIBCO BRL), 2.5mM oligo dT, 5 mM MgCl₂, and 1X PCR buffer consisting of 50 mM KCl, 10mM Tris-HCl, pH 8.3, 1 mM dNTPs, and 1 U/ml RNAse inhibitor. The mixturewas incubated at 42° C. for 1 hr, heated to 99° C. for 5 min to denaturereverse transcriptase, and cooled at 5° C. for 5 min. cDNA from thereverse transcription reaction was subjected to PCR in the presence of0.15 mM and each of 5′ and 3′ primers, 1.25 U of Taq polymerase(Perkin-Elmer), 2 mM MgCl₂, and 1X PCR buffer. PCR was performed in aDNA thermal cycler (Perkin-Elmer) for 35 cycles, each consisting of 95°C. for 1 min and 60° C. for 1 min. After the 35 cycles, there was a timedelay for 7 min at 72° C. The reaction products were visualized by 1.5%agarose gel electrophoresis. The sense and antisense primers,respectively, and the predicted sizes of the RT-PCR reaction productswere as follows:

SF: 5′-CAGCGTTGGGATTCTCAGTAT-3′ (SEQ ID NO:1),5′-CCTATGTTTGTTCGTGTTGGA-3′ (SEQ ID NO:2), 539 bp. c-met:5′-ACAGTGGCATGTCAACATCGCT-3′ (SEQ ID NO:3), 5′-GCTCGGTAGTCTACAGATTC-3′(SEQ ID NO:4), 655 bp. β-actin: 5′-TTGTAACCAACTGGGACGATATGG-3′ (SEQ IDNO:5), 5′-GATCTTGATCTTCATGGTGCTAGG-3′ (SEQ ID NO:6), 764 bp.

The SF primers represent the sense sequence in the K3 domain of theα-chain (nucleotide 979-1000) and the antisense sequence in the 5′portion of the β-chain (nucleotide 1497-1518) of the human SF mRNA.

Nude Mouse Assays. Preparation and Injection of Tumor Cells. Clones tobe studied were grown to 80-90% of confluence in 150 cm² dishes anddetached with trypsin. The trypsin was neutralized with mediumcontaining serum; and the cells were washed twice by centrifugation,counted, and re-suspended in serum-free DMEM at 0.5×10⁸ (Experiment 2)or 1×10⁸/ml (Experiment 3) (see Results). Cells (0.1 ml) were injectedinto 5-6 wk old female nude mice (Frederick Cancer Research andDevelopment Center, Frederick, Md.). Each animal received twoinjections, one on each side, in the mammary fat pads between the firstand second nipples. For experiment 2, ten injections were made perclone, with 5×10⁶ cells per injection; and for experiment 3, 20injections were made per clone, with 1×10⁷ cells per injection.

Measurement of Tumor Size. The animals were ear tagged; and primarytumor growth was assessed by measuring the volume of each tumor atweekly intervals. Three mutually perpendicular diameters (mm) weremeasured using a caliper; and the geometric mean diameter was used tocalculate the tumor volume in mm³ [ie., V=(⅙)π(d₁d₂d₃)]. Only measurabletumors were used to calculate the mean tumor volume for each tumor cellclone at each time point. Animals were sacrificed after 9 (Experiment 2)or 10 wk (Experiment 3), when the largest tumors reached about 20 mm indiameter.

Assessment of Regional Lymph Node and Lung Metastasis. After sacrifice,axillary lymph nodes and both lungs were excised, fixed in formalin,embedded in paraffin, and stained with hematoxylin and eosin (H & E) formicroscopic examination for morphologic evidence of tumor metastasis.Sections were reviewed and scored by a board-certified pathologist (AF).

Preparation of Primary Tumors for Additional Studies. Primary tumorswere excised and weighed; and half of the tumor was fixed in formalin,while the other half was frozen at −80° C. The formalin-fixed tumortissue was embedded in paraffin, and 4 μm sections were cut for theimmunostaining procedures described below. The frozen tissue was used toprepare protein extracts, as described before (Joseph et al., J NatlCancer Inst 87: 372-377 (1995)). Tissues were thawed; washed withextraction buffer [20 mM Tris, pH 7.5, 0.5 M NaCl, 0.1 mM PMSF(phenylmethylsulfonyl fluoride)]; cut into pieces; sonicated in ice-coldextraction buffer (4-6 ml/g of tissue); and clarified by microfuging.The precipitate was re-extracted using the same buffer containing 1 MNaCl. [High salt extractions are used to remove growth factors bound tocell surface and matrix.] The two clarified supernatants were pooled anddialyzed against buffer containing 150 mM NaCl. Protein concentrationsof extracts were determined using the BioRad Coomassie blue dye-bindingmicroassay (BioRad, Richmond, Va.). Samples were stored at −80° C.

In vitro Assays of Tumor Cell Growth and Motility. Proliferation Curves.See Brief Description of FIG. 8.

Plating (Two-Dimensional Colony-Forming) Efficiency (PE). Exponentiallygrowing cultures of MDAMB231 clones were detached with trypsin, and thetrypsin was neutralized with DMEM-10% serum. Cells were counted,serially diluted, and seeded in triplicate at 300 cells per 100 mmdishes in 15 ml of DMEM-10%. Dishes were incubated for 3 wk, after whichthey were stained with crystal violet, and visible colonies (>50 cells)were counted. The PE (in %) was calculated as [(no. ofcolonies/300)×100].

Soft Agar Colony Formation. Anchorage independent growth of MDAMB231clones was assessed using the soft agar assay, as described before(Leone et al., Oncogene 8: 2325-2333 (1993)). Briefly, cells weresuspended at 3.75×10³ cells/ml in DMEM-10% serum with 0.36% Bacto-agar(Difco, Detroit, Mich.). Three thousand cells (0.8 ml of suspension)were spread per well over triplicate wells in 6-well dishes containing ahardened plug of DMEM-10% serum with 0.6% agar. Dishes were fed weeklywith DMEM-10% serum, and colonies were counted microscopically after 4wk of incubation. Colony formation was scored as the number of coloniesof ≧15 cells per 100 colony forming units.

Urokinase (uPA) and Cell Motility Assays. uPA enzyme activity inconditioned media (CM) from MDAMB231 clones (see Table 3 legend) wasassayed by indirect chromogenic substrate (Spectrozyme) assay, as perthe manufacturer's instructions (American Diagnostica, Greenwich,Conn.). High molecular weight human urokinase (80,000 IU/mg) was used asthe standard. uPA titers were normalized by CM protein content. Basalmotility of MDAMB231 clones was assayed using 96-well Boyden chambers,as described below, in the absence of added chemoattractant. The assayincubation interval was 6 hr, and each clone was assayed inquadruplicate.

In vivo Assays of Tumor Cell Proliferation and Death. Proliferating CellNuclear Antigen (PCNA) Index. In vivo tumor cell proliferation wasassessed by immunostaining for PCNA, an antigen selectively expressed incycling cells (Takasaki et al., J Exp Med 154: 1899-1909 (1981)). PCNAwas detected by immunoperoxidase staining of paraffin sections ofprimary tumors with mouse monoclonal anti-human PCNA antibody clonePC-10 (1:200 dilution) (Dako, Carpinteria, Calif.). Each batch of slidesstained included human tonsil tissue as a control containing bothcycling PCNA positive and resting PCNAnegative cells. The PCNA index(percentage of PCNA positive tumor cells) was determined by counting atleast 800 cells per tumor.

Terminal Deoxynucleotidyl Transferase (TdT) Index. In vivo tumor cellapoptosis (programmed cell death) was measured using the recentlydescribed method of TdT in situ labelling (Gavrieli et al., Cell Biol119: 493-501 (1992)). TdT labelling was performed on paraffin sectionsof primary tumors using the In Situ Cell Death Detection Kit fromBoehringer Mannheim, Indianapolis, Ind. The TdT index (percentage ofapoptotic brown TdT positive nuclei) was determined by counting at least1000 cells per tumor.

Angiogenesis-Related Studies. Microvessel Counts (MVCs) of PrimaryTumors. MVCs in regions of the most active tumor angiogenesis(angiogenic “hot spots”) were made by a modification of previouslydescribed methods (Weidner et al., New Engl J Med 324: 1-8 (1991), JNCI84: 1875-1887 (1992)). Paraffin sections were stained with anti-lamininantibody (Gibco, Gaithersburg, Md.) (Wolff et al., Brain Res 604: 79-85(1993)); and vessel profiles were counted in 3-4 different areas oftumor containing the highest microvessel density. Two values weredetermined for each tumor: (a) the peak MVC (single largest number ofmicrovessels per 400× field); and (b) the average peak MVC (mean MVC ofthe 3-4 400× fields with the largest numbers of microvessels). MVCs wereperformed in blinded fashion by a board-certified pathologist (AF).

Microvascular Endothelial Cell Chemotaxis Assays. Assays were performedusing a modification of the method described earlier (Tolsma et al., JCell Biol 122: 497-511 (1993)). HDMECs between passages 5 to 7 weredetached with trypsin; washed three times; counted; resuspended at1.8×10⁶ cells/ml in DMEM containing 0.1% bovine serum albumin(DMEM-BSA); and inoculated at 28 μl (=5×10⁴ cells) per well into thelower wells of a 96-well modified Boyden chamber (Neuroprobe, CabinJohn, Md.). Wells were covered with an 8 μm pore size Nucleopore filterthat was previously coated with 100 μg/ml of Vitrogen in 0.1% aceticacid (Celtrix Laboratories, Palo Alto, Calif.). The chamber wasassembled, inverted, and incubated for 2 hr to allow attachment of cellsto the underside of the filter. The chamber was then re-inverted, and 50μl of test materials in DMEM-BSA were added to the upper wells. Chamberswere incubated for an additional 5 hr, during which time cells migratedagainst gravity from the underside of the filter to the upper side ofthe filter. Non-migrated cells were scraped off the underside of thefilter, and filters were stained using DIFF-QUIK chemicals (Baxter,McGaw Park, Ill.), a differential stain. Migrated cell nuclei werecounted using a calibrated ocular grid as cells per 10 high power (400×)grids.

Rat Cornea Angiogenesis Assay. Angiogenesis was assayed in the avascularrat cornea, as before (Polverini and Leibovich, Lab Invest 51: 635-642(1984)). Briefly, test samples were combined 1:1 with a solution ofHydron (Interferon Laboratories, New Brunswick, N.J.) and air-driedovernight. A 5 μl pellet was inserted into a surgically created pocketin the corneal stroma and placed 1-1.5 mm from the limbus. Corneas wereexamined daily with a dissecting microscope for up to 7 days ofcapillary growth. Responses were scored as positive if sustaineddirectional ingrowth of capillary sprouts and hairpin loops occurredduring the observation period. Responses were scored as negative when noneovascularization was detected or when only an occasional sprout orhairpin loop was observed with no evidence of sustained directionalingrowth. After 7 days, corneas were perfused with colloidal carbon, andwhole mount preparations were examined and photographed.

Measurements of Thrombospondin-1 (TSP1) and Vascular Endothelial CellGrowth Factor (VEGF). The TSP1 content of tumor extracts was measuredusing a specific and sensitive two antibody ELISA similar to thatutilized to measure SF (Joseph et al., J Natl Cancer Inst 87: 372-377(1995)). The first (coating) antibody was anti-human TSP1 mousemonoclonal B7 (1:2000 dilution) (Sigma Chemical Corp., St. Louis, Mo.);and the second (recognizing) antibody was rabbit anti-human TSP1antiserum (1:2000). The assay detected as little as (2.5-5) ng/ml ofTSP1 standard (GIBCO), and did not detect any of a variety of cytokines,growth factors, and extracellular matrix molecules at 500 or 1000 ng/ml.VEGF content of tumor extracts was determined using a specific andsensitive two-antibody VEGF ELISA (Koch et al., J Immunol 152: 4149-4156(1994)).

Statistical Analysis. Values were expressed as means±standard errors ofthe mean (SEMs); and comparisons were made using the two-tailedStudent's t-test. Where appropriate, the chi-squared test was used tocompare proportions.

Expression of Scatter Factor in normal ischemic tissue. In order toascertain potential expression of HGF using the plasmid PRSX24 on normalischemic tissue, Srague-Dawley rats weighing 210-300 were anesthetized,intubated and placed on a positive-pressure respirator. The leftcoronary artery was ligated 3-4 millimeters from its origin to producemycardial infarction, and at the same time, the apices of the heart wereinjected with 40 micrograms of the PRSX24 (HSF) plasmid.

Expression analysis was performed using antihuman HGF monoclonalantibodies by analyzing cross-sectional sections of the apex of rathearts using immunochemistry techniques. Five days following injectionof the plasmid, positive staining was seen in the myocardium whichsupports expression of HSF in the tissue.

II. Results

Transfection and Characterization of Transfected Cell Clones. MDAMB231parent cells were transfected using either: (a) separate vectorscontaining full-length SF cDNA (pRSX24) and the neomycin resistance gene(pSV2neo) (SF+neo); or (b) the neomycin vector pSV2 alone (neo only).Single-cell SF-transfectant (SF+neo) clones and control (neo only)clones were generated by selection in G418. Clones were screened byELISA for SF released into serum-free medium during a 24 hr incubation.Six (SF+neo) clones were found to produce SF titers (9-100 ng/ml) morethan twice as high as high producer MRC5 human lung fibroblasts (4ng/ml). In contrast, none of 20 (neo only) clones produced anydetectable SF. Two high SF-producing (SF+) clones (21 and 29) and twoSF− (neo only) clones (32 and 34) (SF−) that appeared to proliferate atroughly the same rate as the SF+ clones were chosen for further study.

These clones were characterized for expression of SF protein, biologicactivity, and mRNA. SF+ clones 21 and 29 produced high levels ofimmunoreactive SF protein [by ELISA (Table 3) and Western blotting (FIG.6A)] and high levels of SF bioactivity [by MDCK serial dilution scatterassay (Table 3)]. In contrast, SF− clones 32 and 34 produced nodetectable SF by any of these assays. On Northern blotting (FIG. 6B),SF+ clones 21 and 29 exhibited a single mRNA band consistent with thesize of the 2.3 kb SF cDNA; while control clone 34 gave no hybridizingmRNA bands (clone 32 was not tested by Northern analysis). RT-PCRanalysis confirmed that clones 21 and 29 expressed SF mRNA, while clones32 and 34 did not express any detectable SF mRNA (FIG. 7). All fourclones expressed mRNA for the c-met receptor, although mRNA expressionwas slightly lower for clone 32. SF+ clones 21 and 29 also showed astrong in situ hybridization signal using an antisense SF riboprobe,with little or no signal using the corresponding sense riboprobe (datanot shown). These assays indicate high level expression of the SF cDNAin SF+ clones 21 and 29 and no detectable expression in control (neoonly) clones 32 and 34.

In vitro Growth and Motility Characteristics of SF+ vs SF− Cell Clones.In standard two-dimensional proliferation assays, SF+ clones (21 and 29)showed similar growth kinetics to SF− clones (32 and 34) and reachedsimilar saturation densities (FIG. 8). The plating efficiency (PE)(two-dimensional colony formation), is a measure of the proportion ofcells capable of “unlimited proliferation” (defined as formation of avisible, stainable colony) at clonal density. The PE of SF+ clone 21 waslowest (17%); that of SF+ clone 29 and SF− clone 34 were similar (45%and 44%); while SF− clone 32 had an intermediate PE value (30%) (Table3). The average PE values were 31% for SF+ clones vs 37% for SF− clones.Clones 21, 29, and 32 did not form colonies in soft agar during astandard four week assay; while clone 34 formed only a handful ofcolonies. Thus, none of the clones were capable of a significant degreeof anchorage independent growth.

SF is known to induce motility and invasiveness of many human carcinomacell lines (Stoker et al., Nature 327: 238-242 (1987); Rosen et al., InvMetastasis 10: 49-64 (1990), Cancer Res 57: 5315-5322 (1991), Int JCancer 57: 706-714 (1994); Weidner et al., J Cell Biol 111: 2097-2108(1990)). Morphologically, cultures of SF+ cells appeared “scattered”(ie. showed cell dissociation and fibroblastic morphologic changes), ascompared with cultures of SF− cells (data not shown). SF+ clonesproduced higher titers of urokinase, an enzyme associated with cellinvasion, than did SF− clones (Table 3). SF+ clones also showed higherrates of random motility, assayed in chemotaxis chambers in the absenceof exogenous chemoattractant (Table 3). These studies indicate that SF+clones do not exhibit any in vitro growth advantage relative to SF−clones but do exhibit a more motile and invasive phenotype.

In vivo Tumor Growth of SF+ vs SF− Clones in Nude Mice. Tumorigenicityreflects the ability of tumor cells to generate tumors upon injectioninto appropriate animal hosts, and is not the same as primary tumorgrowth. In these studies, the parent cell line (MDAMB231), obtained fromthe ATCC, had not been pre-adapted or selected for in vivo tumor growth.In a pilot study (Experiment 1), injection of 4×10⁵ cells yielded atumor take rate (judged after 3 wk) of 8/20 (40%) for SF+ clones (21+29)and 3/20 (15%) for SF− clones (32+34). In Experiment 2, 5×10⁶ cells wereinjected and tumor take rates were 16/18 (89%) for clones (21+29) and9/18 (50%) for clones (32+34). In Experiment 3, 10×10⁶ cells wereinjected, and the tumor take rates were 39/40 (98%) for clones (21+29)and 36/38 (95%) for clones (32+34). These findings suggest that the SF−clones are “not quite tumorigenic” in terms of their in vivo growth, andboth SF+ and SF− cells require large initial innocula [(5-10)×10⁶ cellsper injection] to generate tumors.

Primary Tumor Growth. Tumors generated from SF+ clones (21 and 29)showed significantly increased growth rates in all three experiments, ascompared with tumors from SF− control clones (32 and 34). Tumor growthcurves from two experiments are shown in FIG. 9A (Experiment 3) and FIG.9B (Experiment 2). Experiments were carried out for 9-10 weeks, by whichtime the largest tumors had reached about 2 cm in diameter. In oneexperiment (FIG. 9A), SF+ clone 29 tumors showed similar growth to theSF− tumors until week 5, after which it overtook both SF− clones. In theother experiment (FIG. 9B), both SF+ clone tumors were consistentlylarger than both SF− clone tumors. At sacrifice, the weights of SF+tumors were significantly greater than those of SF− tumors (P<0.001), bya ratio similar to that of the primary tumor volumes (Table 4). Thus,while caliper measurements of tumor size may include skin tissue, thesemeasurements are still likely to reflect tumor size.

Regional and Metastatic Tumor Dissemination. To study tumordissemination, H & E stained paraffin sections of axillary lymph nodesand lungs were examined for morphologic evidence of tumor cells by lightmicroscopy. SF+ clones showed a higher proportion of lymph nodepositivity (37%) than did SF− clones (13%) (P=0.011, chi-squared test)(Table 4). On the other hand, neither SF+ nor SF− clones gave rise tolung metastases. The presence of occasional isolated tumor cells in someof the lung samples cannot be ruled out, it was generally easy toobserve a small cluster of tumor cells in the murine nodes. Inrepresentative nodes scored as positive by tumor cell morphology,immunochemical staining with human pre-keratin antibody (which stainsadenocarcinoma) revealed an intense positive ring of cytoplasmicstaining within the tumor cells (data not shown).

Additional Studies. Thus, SF+ clones grew more rapidly as tumors andshowed a higher rate of lymph node metastasis than did SF− clones,despite the lack of an in vitro proliferative advantage for the SF+cells. Additional studies were performed to investigate the discrepancybetween these in vivo and in vitro results.

Expression of SF in vivo in Tumors. To confirm that SF+ and SF− clonesmaintained their respective SF producing phenotypes in vivo, tumors wereexcised after the animals were sacrificed, and protein extracts wereprepared and assayed for SF by ELISA. These studies revealed thatextracts of SF+ clone (21+29) tumors had about 50-fold higher SF contentthan did extracts of SF− clone (32+34) tumors (43 vs 0.9 ng SF/mgprotein), confirming high level SF production in vivo in SF+ tumors(Table 5). Secondly, five different SF+ clone 21 tumors and five SF+clone 29 tumors, were explanted into cell culture, selected in G418 tokill host stroma, passaged several times to obtain what appeared to bepure tumor cell cultures, and tested for release of SF into serum-freeculture medium. The SF+ clones all produced very high SF titers, similarto those of primary clones that had not been through in vivo passage.One of these secondary clones (SF+ clone 29-1L) was re-injected intonude mice and exhibited in vivo growth as fast as or faster than primarySF+ clone 21 to clone 29 (data not shown).

PCNA Index. PCNA is an antigen expressed selectively in cycling cells,during the late G1 to M phase (Takasaki et al., J Exp Med 154: 1899-1909(1981)). The PCNA index (percentage of PCNA positive cells detected byimmunohistochemical staining) is a measure of the percentage of cyclingcells within a tissue. To determine if SF+ tumors have a higherproportion of cycling cells than SF− tumors, paraffin-embedded primarytumor sections were immunostained with anti-PCNA antibody. A modest butsignificant increase in the PCNA index of SF+ vs SF− tumors was found(70% vs 60%, P<0.01) (Table 6).

TdT Labeling Index. TdT in situ labeling is a recently described methodby which apoptosis can be assessed in tissue sections (Gavrieli et al.,J Cell Biol 119: 493-501 (1992)). Apoptosis in normal tissues and tumorsmay have a major impact on steady-state maintenance and on growth rates(Wyllie, Cancer Metastas Rev 11: 95-103 (1992); Holmgren et al., NatureMedicine 1: 149-153 (1995)). Thus, increased rates of apoptosis in SF−relative to SF+ tumors could contribute to more rapid overall growth ofthe latter. TdT labeling of primary tumor sections was performed and thepercentage of apoptotic single cells within the mass of surroundingviable cells (TdT labeling index) was determined. This analysis revealedthat the TdT indices were similar in SF+ vs SF− tumors, and theseindices were very low for all four clones (ca. 1%) (Table 6). Thus, itis unlikely that differential rates of apoptosis can explain observeddifference in tumor growth.

Angiogenesis-Related Studies. Microvessel Counts (MVCs) of PrimaryTumors. Recent studies suggest that MVCs of paraffin sections of humanbreast cancers and other tumor types can be utilized as a measure oftumor angiogenesis, and that these MVC measurements are independentmarkers of prognosis (Weidner et al., New Engl J Med 324: 1-8 (1991),JNCI 84: 1875-1887 (1992)). The MVC method involves counting the numberof immunostained microvessel profiles per unit area in the region oftumor containing the most active tumor angiogenesis and thus reflectsvessel formation in “angiogenic hot spots”. MVCs in SF+ vs SF− primarytumors were measured as both the highest individual MVC (“peak MVC”) andthe average MVC of 3-4 fields with the highest vessel densities(“average peak MVC”). It was found that SF+ tumors (21 and 29) had60-70% higher peak and average peak MVCs as compared with SF− tumors (32and 34) (Table 7). Statistical comparison of pooled MVC results forclones (21+29) vs clones (32+34) revealed P<0.001 for both peak MVC andaverage peak MVC (two-tailed t-test). Thus, using MVC as the criterion,SF+ tumors exhibited more tumor angiogenesis than did SF− tumors.

Microvascular Endothelial Cell Chemotaxis Assays. Chemotactic activityfor microvascular endothelial cells is an in vitro correlate of in vivoangiogenic activity (Tolsma et al., J Cell Biol 122: 497-511 (1993)). Indifferent batches of HDMEC cells, rhSF gave (2.5-10)-fold stimulation ofchemotaxis, with maximal responses at 5-10 ng/ml and half-maximalresponses at about 0.5 ng/ml of SF. In the presence of 20 μg/ml ofanti-SF neutralizing monoclonal MO294 (R & D Systems), more than 90% ofthe SF− stimulated chemotactic migration was reproducibly inhibited at20 ng/ml of SF and 80-85 % of the stimulated migration was inhibited at100 ng/ml of SF (FIG. 10A). Conditioned medium (CM) from SF+ culturedmammary carcinoma clones 21 and 29 (normalized by secreted proteincontent) gave dose-dependent stimulation of chemotaxis of HDMECs,whereas CM from control SF− clones 32 and 34 gave little or nostimulation of HDMEC chemotaxis (FIG. 10B). All of the stimulatedchemotactic migration induced by clone 21 and 29 CM was blocked byMO294, which had no effect on migration in the presence of clone 32 and34 CM (FIG. 10C). These findings indicate that SF-transfection enhancesthe net angiogenic balance of the cultured mammary carcinoma cells.

To assess the angiogenic phenotype of the actual tumors, proteinextracts form tumors excised at the end of the experiment were assayedfor HDMEC chemotactic activity. Extracts from clones 21 and 29 showeddefinite chemotactic activity, while extracts from clones 32 and 34showed no activity (FIG. 10D). In tests of other extracts at 50 μg/ml,SF+ tumors reproducibly gave greater than control chemotactic migration,while SF− tumors always gave less than control migration. The latterfinding might be due to the presence of an inhibitor or to toxicity ofthe extract. The enhanced chemotactic activity in 50 μg/ml of clone 21and 29 tumor extracts was neutralized to below control levels (about 75%of control) by anti-SF monoclonal (FIG. 5E). Again, this observation mayreflect the presence of an inhibitor or toxin in the extractpreparation.

Rat Corneal Neovascularization Assay. The rat cornea assay (27) was usedto assess in vivo angiogenic activity in tumor extracts (Table 8 andFIGS. 11A-11E). As observed before (15), recombinant human SF, apositive control, gave a positive neovascular response (FIG. 11B) thatwas blocked by neutralizing anti-SF antibody (Table 8). Extracts from anSF+ clone 21 tumor also gave strong positive responses (FIG. 11C) thatwere inhibited by anti-SF antibody (FIG. 11E). On the other hand,extracts from an SF− clone 32 tumor yielded weakly positive responses(FIG. 11D) that were not inhibited by anti-SF antibody. Thus, under theconditions of this assay, extracts from both SF-transfected and controltumors exhibited detectable angiogenic activity, but the SF+ transfectedtumors had higher levels of angiogenic activity than did the controltransfected tumors.

VEGF and TSP1 Assays of Extracts of Primary Tumors. To determine ifexpression of angiogenic regulatory molecules other than SF differed inSF+ vs SF− tumors, levels of the angiogenic growth factor VEGF and theanti-angiogenic macromolecule TSP1 were measured in tumor extracts. EachSF− tumor clone (32 and 34) had higher VEGF content than each SF+ clone(21 and 29) (P<0.001) (Table 5). However, the observed titers of VEGF(0.1-0.5 ng/mg protein) were two orders of magnitude lower than SFtiters in SF+ tumors (40 ng/mg protein). CM was also tested from thecultured cell clones and found similar results: clones 32 and 34 CMcontained more immunoreactive VEGF by Western blotting than did clone 21and 29 CM (data not shown). Assays of TSP1 revealed that the mostrapidly growing tumor (SF+ clone 29) had the lowest TSP1 content, whilethe slowest growing tumor (SF− clone 32) had the highest TSP1 content(P<0.001, clone 29 vs 32) (Table 5). However, clone 21 (SF+) and 34(SF−) tumors had similar, intermediate levels of TSP1.

TABLE 3 In Vitro Characteristics of Scatter Factor (SF)-Transfected andControl Clones SF Production Soft agar μPA Productions Migration Clone(ng/ml) (units/ml) PE (%) colonies (lU/mg protein) (cells/10 grids) 21(SF + neo) 56 128-256 17 ± 0 0 283 362 ± 8 2g (SF + neo) 43 128-256 45 ±2 0 501 377 ± 9 32 (neo) 0 0 30 ± 1 0 123 174 ± 5 34 (neo) 0 0 44 ± 3 289 190 ± 5 PE = Plating efficiency: μPA = Urokinase. # To measure SFproduction, confluent cultures were incubated in serum-free OMEM (0.1ml/cm²) for 24 hours, and the conditioned media (CM) were assayed forimmunocreative SF by ELISA (ng/ml) and for SF bioactivity using the MOCKserial dilution scatter assay (unit/ml). PE is the percentage of cellsthat form visible, stainable two-dimensional colonies after 3 weeks ofincubation: values listed are means ± SEM of triplicate determinations.# Soft agar colony formation is the number of three-dimensional coloniesof ≧ 15 cells per 100 colony forming units after 4 weeks. μPA enzymeactivity was measured in CM and normalized per mg of CM protein.Migration values were measured in 96-watt modified Boyden chambers inthe absence of chemoattractant and represent means ± SEM ofquadruplicate assays.

TABLE 4 Primary Tumor Size, Lymph Node Status, and Lung Metastases atTime of Killing Lung Primary tumor Primary tumor Lymph nodes metastasesClone weight (g) volume (mm³) pos/total (%) pos/total (%) 21 0.76 ± 0.11(25) 723 ± 93 (25) 6/24 (25%) 0 29 1.01 ± 0.24 (17) 866 ± 193 (17) 9/17(63) 0 32 0.19 ± 0.05 (16) 121 ± 22 (16) 2/15 (13) 0 34 0.36 ± 0.08 (16)321 ± 67 (16) 2/15 (13) 0 Weights and volumes aee expressed as means ±SEM (number of tumors assayed). Statistical comparions of pooled datafor SF + tumors (clone 21 + 29) versus SF - tumors (clone 32 + 34):tumor weight p < 0.001 (two-tailed t-test); tumor volume, p < 0.001(two-tailed t-test); lymph node positivity, = 0.011 (chi-squared test).Pos = Positive.

TABLE 5 Content of Angiogenesis Regulatory Factors in Primary TumorExtracts¹. SF Content VEGF Content TSP1 Content Clone (ng SF/mg protein)(ng/mg protein) (ng TSP1/mg protein) 21 39.3 ± 3.2 (25) 0.13 ± 0.02 (4)485 ± 68 (17) 29 47.4 ± 3.8 (17) 0.17 ± 0.03 (4) 283 ± 67 (13) 32  1.1 ±0.3 (16) 0.51 ± 0.14 (4) 1396 ± 330 (14) 34  0.8 ± 0.3 (16) 0.48 ± 0.10(4) 481 ± 81 (11) ¹Abbreviations: SF = scatter factor, VEGF = vascularendothelial growth factor; TSP1 = thrombospondin-1. Primary tumorstested were from Experiments 2 + 3 for SF and Experiment 3 for VEGF andTSP1. Values listed are means ± SEMs (number of tumors assayed). # ForSF, statistical comparison of pooled clones (21 + 29) vs (32 + 34) gaveP < 0.001. For VEGF, comparison of pooled clones (21 + 29) vs (32 + 34)gave P < 0.001. For TSP1, values for clone 32 were significantly greaterthan those for clones 21 (P < 0.01), 29 (P <0.001), and 34 (P = 0.027).TSP1 values for clone 29 were lower than those for clones 21 (P =0.046), 32 (P < 0.001), and 34 (P = 0.07).

TABLE 6 Cell Proliferation (PCNA Index), and Cell Death (TdT Index) InVivo in Primary Tumors¹. PCNA Index TdT Labeling Index Clone (% PositiveCells) (% Positive Cells) 21 69 ± 2 (19) 0.8 ± 0.1 (22) 29 71 ± 2 (13)1.0 ± 0.1 (16) 32 57 ± 6 (14) 1.0 ± 0.2 (16) 34 63 ± 4 (12) 0.9 ± 0.1(12) ¹Abbreviations: PCNA = proliferating cell nuclear antigen; TdT =terminal deoxytansferase. Values listed are means ± SEMS (number oftumors assayed). PCNA and TdT indices were determined from staining ofparaffin sections of tumors. Staining and counting procedures aredescribed in Materials and Methods. For TdT, values were derived fromExperiments 2 + 3; for PCNA, values were derived from Experiment 3.Statistical comparison of pooled clones (21 + 29) vs (32 + 34) gave: P<0.001 (SF content); P = 0.006 (PCNA index); and P >0.1 (TdT index).

TABLE 7 Microvessel Counts (MVCs) of Primary Tumors¹. Average Clone PeakMVC Peak MVC 21 6.4 ± 0.4 (23) 4.7 ± 0.3 (23) 29 6.4 ± 0.8 (17) 4.6 ±0.5 (17) 32 4.1 ± 0.5 (15) 3.2 ± 0.4 (15) 34 3.5 ± 0.5 (15) 2.5 ± 0.3(15) ¹Tumor sections were stained and examined for microvessels asdescribed in Materials and Methods. Peak MVC is the number ofmicrovessels per field (40× objective, 10× ocular) in the region of mostactive tumor angiogenes. Average peak MVC is the mean MVC of the 3Afields with the highest microvessel count. Values listed are means ±SEMs (number of tumors assayed). Comparisons of pooled clones (21 + 29)vs (32 + 34) gave P <0.001 (peak MVC) and P <0.001 (average peak MVC).

TABLE 8 Angiogenic Responses Induced by SF-Transfected and Control HumanBreast Cancer Extracts Corneal neovascularization proportion of positiveTest sample responses (%) Controls Hydron + PBS 0/4  (0) SF (50 ng) 4/4(100) M0294 (500 ng) 0/3  (0) SF (50 ng) + M0294 (2.5 0/3  (0) μg) Tumorextracts ± M0294 SF+ Clone 21 (5 μg) 4/4 (100) SF+ Clone 21 + M0294 1/4 (25) SF− Clone 32 (5 μg)  1/3*  (33) SF− Clone 32 + M0294  1/4*  (25)SF = Recombinant human scatter factor; M0294 = Anti-SF− (neutralizingmonoclonal. *Assay methods and response criteria are described in“Materials and Methods. Neavascular responses induced by SF− tumorextracts with or without M0294 were substantially weaker in intensitythan responses induced by SF+ extracts.

III. Discussion

The inventors have shown that transfection of SF cDNA into the humanbreast cancer cell line MDAMB231 results in an increased rate oforthotopic tumor growth in nude mice of tumors derived from SF+ cellclones as compared with SF− (control) cell clones. Tumors derived fromSF+ clones also had significantly higher rates of spread to regionallymph nodes; but neither SF+ nor SF− clones gave any detectablepulmonary metastases. The SF+ clones overexpressed SF mRNA andoverproduced SF protein in vitro in cell culture and in vivo in tumors.In contrast, SF− clones produced little or no SF in vitro or in vivo.Thus, it is unlikely that the lack of SF production by SF− controlclones and by parental MDAMB231 cells was due to absence of a requiredfactor(s) in the cell culture environment that is present in the in vivoenvironment. Moreover, SF+ clones did not show any in vitro growthadvantage over SF− clones, as indicated by assays of proliferationkinetics, plating efficiency, and soft agar colony formation.

SF+ clones showed scattering, increased in vitro cell motility, andincreased urokinase production as compared with SF− clones. Thesefindings are consistent with the observation that MDAMB231 cells expressthe c-met receptor and with many previous observations that SFup-regulates the motile and invasive phenotype of epithelial andcarcinoma cells (Stoker et al., Nature 327: 238-242 (1987); Rosen etal., Inv Metastasis 10: 49-64 (1990), Cancer Res 57: 5315-5321 (1991),Int J Cancer 57: 706-714 (1994); Weidner et al., J Cell Biol 111:2097-2108 (1990)). In a prior study of mouse 3T3 cells, it was shownthat transfection of human SF plus human c-met cDNAs induced a highlytumorigenic phenotype via an autocrine loop (Rong et al., Mol Cell Biol12: 5152-5158 (1992)). A similar autocrine loop may have contributed tothe increased tumorigenicity and to the increased rate of disseminationto axillary lymph nodes of SF+ human breast cancer clones relative toSF− clones. However, it is unlikely that autocrine stimulation per secontributed to the increased growth rate of SF+ tumors, since SF+ andSF− cells exhibited similar in vitro proliferation kinetics. Sinceaxillary nodal metastasis is also a function of primary tumor size, itis not certain the degree to which increased tumor cell invasiveness asopposed to increased primary tumor size were responsible for the higherrate of axillary nodal involvement of SF+ tumor cells.

The more rapid in vivo growth of SF+ tumors may be explained, in part,by an increase in the proportion of cycling cells (PCNA index), but thedifference in the PCNA indices of SF+ vs SF− tumors was modest (70% vs60%). The in vivo cell cycle times were not measured, which might havebeen shorter in the SF+ tumor group. Slower tumor growth or tumor stasishas been attributable to increased levels of apoptosis in some in vivotumor models (Holmgren et al., Nature Medicine 1: 149-153 (1995)). Thedifference in in vivo growth rates of SF+ vs SF− tumors cannot beexplained by differences in the rates of apoptosis, since both tumortypes had equally low (ca. 1%) TdT labeling indices. Classic regions ofnecrosis were observed in both SF+ and SF− tumors, but there did notappear to be obvious differences in the degrees of necrosis either.However, several lines of evidence suggest that SF+ tumors had anenhanced angiogenic phenotype as compared with SF− tumors.

Microvessel density in highly angiogenic regions of human tumors wasproposed as a standard measure of tumor angiogenesis and as a prognosticindicator (Weidner et al., New Engl J Med 324: 1-8 (1991), JNCI 84:1875-1887 (1992)). Sections of SF+ tumors had higher microvesseldensities than SF− tumors, whether the single highest MVC value wastaken or the 3-4 highest values were averaged (P<0.001). Chemotacticactivity for capillary endothelial cells is thought to be an in vitrocorrelate of angiogenesis (Tolsma et al., J Cell Biol 122: 497-511(1993)). The inventors found that: (a) CM from SF+ cells and proteinextracts from SF+ primary tumors had increased chemotactic activity forHDMECs as compared with CM and extracts from SF− cells and tumors; and(b) the chemotactic activity from SF+ cells and tumors was neutralizedby an anti-SF monoclonal. SF+ tumor extracts also had more in vivoangiogenic activity in the rat corneal assay than did SF− tumorextracts; and the angiogenic activity from SF+ extracts was markedlyinhibited by the anti-SF monoclonal. The observations that CM andextracts from SF+ tumors have increased chemotactic and angiogenicactivity are consistent with the results from the microvessel counts.These findings suggest that higher levels of tumor angiogenesiscontributed to the enhanced growth of SF+ tumors.

To determine if secondary alterations of other angiogenic regulatorscould have contributed to the observed results, the tumor contents ofthe angiogenic growth factor VEGF and the anti-angiogenic glycoproteinTSP1 were measured. The VEGF content was higher in SF− tumors than inSF+ tumors; and the VEGF titers were 100-fold lower than the SF titersin SF+ tumors. Thus, it is unlikely that alterations in VEGF expressioncontributed to increased tumor angiogenesis in SF+ tumors. TSP1 is amultidomain adhesive glycoprotein of the extracellular matrix withpotent anti-angiogenic activity in vitro and in vivo (Good et al., PNASUSA 87: 6624-6628 (1990); Tolsma et al., J Cell Biol 122: 497-511(1993)). Transfection of MDAMB435 human breast cancer cells withfull-length TSP1 cDNA caused reduced growth of the primary tumor,reduced metastatic rate, and inhibition of angiogenesis (Weinstat-Saslowet al., Cancer Res 54: 6504-6511 (1994)). Assays of the TSP1 content oftumor extracts revealed that clone 29 SF+ tumors, the fastest growing ofthe four clones studied, had the lowest TSP1 content, while clone 32tumors, the slowest growing, had the highest TSP1 content (P<0.001 forclone 29 vs 32). The net angiogenic phenotype of a tumor is likely to bedetermined by the balance between angiogenic and anti-angiogenic factorswithin the tumor. Thus, the finding of an inverse relationship betweenTSP1 content and tumor growth rate for clones 29 and 32 suggest thatTSP1 may have influenced the angiogenic phenotype of these clones.However, SF+ clone 21 tumors grew more rapidly, showed higher MVCs, andcontained more angiogenic activity than SF− clone 34 tumors, despitehaving similar TSP1 content. Therefore, it does not appear as ifsecondary alteration in tumoral TSP1 content was a major determinant oftumor growth or angiogenesis in this study.

The inventors' studies indicate that endogenous overexpression of SFstimulates growth of human breast cancer xenografts, but they do not bythemselves establish a role for SF in malignant growth of breast cancer.Various other studies provide circumstantial evidence that SFcontributes to breast cancer growth or progression. While none of 10lines of human breast carcinoma cells produced SF in vitro (Yamashita etal., Res Commun Chem Pathol Pharmacol 82: 249-252 (1993); Rosen et al.,J Cell Biol 127: 225-234 (1994)), both SF and c-met receptor weredetected in vivo in carcinoma cells by in situ hybridization andimmunostaining of human breast cancer tissue sections (Wang et al.,Science 266: 117-119 (1994); Tuck et al., Am J Pathol 148: 225-232(1996)). Thus, carcinoma cells may lose the ability to produce SF duringin vitro passage, or the cells that give rise to mass cultures may be SFnegative. Yamashita and colleagues (1994) measured the SF content of 258primary invasive breast cancers and reported that high SF content is apowerful, independent predictor of relapse and death. The SF content of167 primary breast cancers was measured and a much higher SF content wasfound in invasive vs ductal carcinoma-in-situ tumors and in primarytumors in which axially lymph nodes were involved (Yao et al., inpress). It was found that tumor SF content was strongly associated withtumor content of von Willebrand's factor, suggesting a correlationbetween SF content and tumor vascularity (34). The highest SF contentvalues in human breast cancers (3-14 ng SF/mg protein) were less thanthe average values in SF+ xenografts (43 ng SF/mg protein). However, theminimum SF level needed to stimulate tumor growth is not known. Inaddition, human breast stroma (e.g., endothelial cells) may be moresensitive to human SF than is mouse mammary fat pad stroma. Takentogether with other studies, the findings set forth herein strengthenthe case that SF contributes to human breast cancer growth.

Angiogenic regulatory factors have been found to modulate growth ofhuman breast cancers in several other orthotopic xenograft models. Ascited above, transfection of MDAMB435 cells with TSP1 inhibited tumorgrowth and angiogenesis (Weinstat-Saslow et al., Cancer Res 54:6504-6511 (1994)). MCF-7 cells, which normally require estrogensupplementation for sustained in vivo tumor growth in nude mice,exhibited progressive estrogen-independent tumor growth whenpro-angiogenic fibroblast growth factors (FGF-1 and FGF-4) weretransfected into and overexpressed in the cells. These FGF-dependenttumors continued to grow, even in the presence of tamoxifen (Kern etal., Breast Cancer Res Treat 31: 153-165 (1994)). However, treatment ofuntransfected MCF-7 tumors with tamoxifen resulted in tumor regressionand necrosis, which could be attributed, in part, to inhibition ofangiogenesis and of endothelium growth (Haran et al., Cancer Res 54:5511-5514 (1994)). Systemic administration of angiostatin, a fragment ofplasminogen with strong anti-angiogenic activity, inhibited the growthof a subcutaneous human breast carcinoma primary tumor in SCID mice(O'Reilly et al., Nature Med 2: 689-692 (1996)). Angiostatin-treatedtumors showed reduced angiogenesis and increased tumor cell apoptoticindices in the absence of any change in the tumor cell proliferativeindex.

In conclusion, transfection with SF cDNA induces increased growth ofhuman breast cancer cells in the mammary fat pads of nude mice. Theincreased growth rate of SF-transfected tumors is due, in part, toincreased tumor angiogenesis induced by SF.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of various aspects of the invention. Thus, it isto be understood that numerous modifications may be made in theillustrative embodiments and other arrangements may be devised withoutdeparting from the spirit and scope of the invention.

6 21 nucleic acid single linear other nucleic acid <Unknown> No 1CAGCGTTGGG ATTCTCAGTA T 21 21 nucleic acid single linear other nucleicacid <Unknown> No 2 CCTATGTTTG TTCGTGTTGG A 21 22 nucleic acid singlelinear other nucleic acid <Unknown> No 3 ACAGTGGCAT GTCAACATCG CT 22 20nucleic acid single linear other nucleic acid <Unknown> No 4 GCTCGGTAGTCTACAGATTC 20 24 nucleic acid single linear other nucleic acid <Unknown>No 5 TTGTAACCAA CTGGGACGAT ATGG 24 24 nucleic acid single linear othernucleic acid <Unknown> No 6 GATCTTGATC TTCATGGTGC TAGG 24

What is claimed is:
 1. A method for reducing angiogenesis caused byscatter factor in a subject, the method comprising local administrationto the subject an anti-scatter factor antibody effective to reduceangiogenesis caused by scatter factor in said subject.